HUT51144A - Process for production of medical compositions containing peptid or protein active substances and alpha amin boric acid - Google Patents

Abstract

Pharmaceutical compositions of peptide or protein drugs are provided which contain α-­aminoboronic acid derivatives to stabilize these drugs during their absorption through various mucosae, such as nasal and buccal mucosae, and eventually improve their systemic bioavailability.

Description

FIELD OF THE INVENTION The present invention relates to a process for the preparation of pharmaceutical compositions containing a peptide or protein active ingredient and / or aminoboronic acid derivatives, more particularly to compositions which can be administered by absorption through various mucous membranes.

Peptides and proteins are usually administered parenterally because they have low bioavailability when administered by other means, such as oral, nasal, oral, or vaginal. The proportion of peptide or protein that is absorbed intact into the systemic circulation depends on the degree of resistance of the peptide or protein to the degradation action of peptidases and proteases, which are commonly found at the site of administration and also in mucosal barriers.

Peptidases are generally responsible for the degradation of peptides in animal tissues (Mammalian Proteases, and

Publishers: A.J. Barratt 0. K. McDonald, II. Volume 23-102).

Enzyme inhibitors are considered to be very effective in the degradation of orally administered peptides, resulting in poor bioavailability of orally administered peptides. However, this barrier is considered to be moderately effective in non-oral modes of administration and therefore these non-oral modes of administration are beneficial for the bioavailability of peptides. For example, the bioavailability of one of the most extensively studied peptides, leuprolide, was found to be three-fold, 35-fold and 112-fold higher when administered by nasal, rectal and vaginal administration, respectively. However, the proportion of the peptide absorbed into the systemic circulation is only 0.1, 1.2, and

3.8 Va for the delivered dose. On the other hand, more than 65% of the administered dose is absorbed systemically by subcutaneous administration of leupro lid / Okada et al., 0. Pharm. Sci. 71: 1367-1371 (1982) 7. It is possible that these results are a consequence of the action of aminopeptidases present in the mucosa of the nose, rectum and vagina / Stratford et al., Int. J. of Pharm. 30, 73-82 (1985) 7-

Hydrolysis of 1euciπ-enkepha 1 in the nasal cavity is suggested to be one of the possible factors for the low bioavailability of peptides administered nasally. Hussain, J. Faraj, A. Yukihiko and J. E. Truelove, Biochem. Biophys. Gap. Com., 133, 923 (1985) 7.

Transient analogue inhibitors are molecules that are similar in structure to the substrate in its transition state, but have a significantly higher affinity for the active site of the enzyme than the substrate itself. Such inhibitors are the amino boronic acid derivatives disclosed in U.S. Patent Nos. 4,537,773 and 4,499,082, which are potent, reversible inhibitors of certain proteolytic enzymes. These amino boronic acid derivatives are tetrahedral borate species of trigonal boric acid. The tetrahedral borates are similar to the transition state during peptide hydrolysis by proteases.

There is a need for effective inhibitors of aminopeptidases that improve the delivery of pharmacologically active peptides and proteins by nasal, oral, rectal and intramuscular administration. The amino boronic acid derivatives satisfy this need.

The pharmaceutical compositions of the present invention consist essentially of a pharmaceutically acceptable carrier, a protein or peptide, and optionally an absorption enhancer and a 't-aminoboronic acid derivative of formula (I). In the general formula (I)

- HW represents mineral acid, sulfonic acid, alkanoic acid or perfluoroalkanoic acid;

- Y is a group of a dihydroxy compound having at least two hydroxy groups separated by at least two linked chain or ring atoms from the carbon atoms of said chain or ring and optionally from a nitrogen, sulfur or oxygen heteroatom They are,

- R is alkyl or -CH ₂ R ^X ;

2

- R is -XR, aryl or aryl substituted with one or more alkyl groups;

- X is oxygen or sulfur;

R is hydrogen, alkyl or

-Si R ³ R ⁴ R ⁵ ;

• · · · 4 • · 4 · ·· «,

- 5 3 4 5 - R, R and R are independently alkyl, aryl or aryl substituted with one or more alkoxy groups.

These potent inhibitors of aminopeptidases stabilize and improve the transport of pharmacologically active peptides and proteins administered nasally, orally, rectally, and intramuscularly.

The invention will now be described in more detail.

1) The term protein or peptide specifically refers to peptides which are sensitive to proteases, in particular aminopeptidases, which bridge them. Preferred proteins or peptides are leucine-enkephalin, LH-RH, Tyr-Phe-Met-Arg-Phe-NH 4, Tyr-Bradykinin and Thymopoietin II (TP5).

2) The term "absorption enhancer" refers to substances that enhance the uptake of proteins and peptides and can be used to alter the penetration characteristics of active ingredients. Preferred absorption enhancers are through sodium cholate, sodium taurocholate, sodium glycocholate, sodium deoxycholate, sodium taurodeoxycholate and sodium glicodeoxychol. Other absorption enhancers equivalent to these are known to those skilled in the art.

3) A carrier suitable for medical purposes · · · · · · · · · · · · · · · · · · · ·

6 denotes any non-toxic pharmaceutically acceptable carrier. Suitable carriers include, for example, capsules, tablets, suppositories, nasal sprays, mucosal patches and intrauterine devices, liquid solutions or suspensions. The components may also be dissolved individually or together in carriers conventionally used for injection. The formulations may be formulated in a manner known to those skilled in the art. Formulations suitable for other routes of administration may be formulated with conventional non-toxic ingredients known in the art. Preferably, a carrier suitable for administration by absorption through the mucosa is used.

4) The term X-aminoboronic acid derivative means all derivatives described in U.S. Patent Nos. 4,499,082 and 4,537,773. Preferred are the derivatives described in Examples 5, 13 and 21 of U.S. Patent 4,537,773.

The t-aminoboronic acid derivatives useful in the process of the invention significantly reduce peptides and proteins at nanomolar to micromolar concentrations, such as leucine-enkephalin, LH-RH, Tyr-Phe-Met-Arg-Phe-N1- and Tyr- Hydrolysis of Bradykinin, while other known inhibitors, such as Bestatin and Puromycin, exhibit only much higher concentrations of similar inhibitory activity.

··········•································································································································································ Managed: The following information is provided by the user:

As mentioned above, the transport of peptides and proteins in the presence of derivatives of aminoboronic acid may be accomplished by several different routes. The derivative is used in an effective amount to inhibit the hydrolysis of the peptide or protein by proteases and in an amount sufficient to enhance bioavailability through various mucous membranes.

In the following studies on peptides

Transport without solutions of X-aminoboronic acid derivative and, if desired, the use of solutions, as well as an absorption enhancer, are investigated. Formulations for nasal administration are generally liquid compositions containing the peptides together with an inhibitor of hydrolysis and optionally an absorption enhancer. Typically, these formulations are prepared so that about 0.1 mL of liquid, administered as a metered spray, contains all of the ingredients.

The proportion of ingredients is generally 0.0001 to 10% by weight peptide or protein, 0-10% by weight enhancer, 0.00001-5% by weight << - aminoboronic acid derivative inhibitor, 0-10% by weight preservative, additional amount distilled water, saline or buffer.

Typical formulations for use in the oral cavity are 0.0001 to 10% by weight of a peptide or protein, 0 to 10% by weight absorption aid, 0.00001 to 5% by weight inhibitor, 0 to 10% by weight preservative, 0 to 99.9% by weight. · ··· ·

- containing 8 non-toxic carriers, 0 to 20% suspending agent and additionally containing 100% polymeric matrix and / or mucoadhesive material.

Typical oral formulations containing the peptide or protein active ingredient, inhibitor and optionally an absorption enhancer may further comprise a diluent, granulating agent, preservative, binder, flavoring agent, coloring agent or coating agent.

Other formulations, such as suppositories and intrauterine devices, may also be prepared by known methods and known excipients known to those skilled in the art.

The invention is illustrated by the following examples.

Animal experiments are conducted to investigate the efficacy of the aminoboronic acid derivative inhibitors of the present invention. Hussain, J. Furaj, A. Yukihiko and J. E. Truelove, Biochem. Biophys. Gap. Com. 133, 923 (1985) 7 to measure the degradation of proteins and peptides in situ and in vivo in the nasal cavity of rats.

Assays were performed on Sprague-Dawley rats weighing approximately 300 g. Each rat was anesthetized with 50 mg / kg sodium pentobarbitol. An incision was made in their neck and cannulae of PE 240 tubes were inserted into their trachea. For in situ testing, another tube is inserted through the esophagus into the back of the nasal cavity. «« · · · · · · · · · · · · · · · · ·

9 through which the perfusion solution is introduced. The nasal palate is closed with a patch to prevent the perfusion solution from dripping from the nose into the oral cavity. For in vivo in situ studies, a cannula consisting of a closed PE 240 tube was placed in one end of the esophagus. Press the closed end of the tube to the posterior end of the nasal passage and fix it there. The nasal palate is closed with a patch to prevent the perfusion solution from passing from the nose to the oral cavity. 50 µl peptide solution was administered to one nostril of rats in two forms, the control solution did not contain any added inhibitor, and the test solution contained the amount of inhibitor indicated in the examples. The peptide addition solution is 0.1 M phosphate buffer pH 7.4. At appropriate intervals (at one time in a rat), the active ingredient is washed through the other nostril using 10 mL of 0.1 M phosphate buffer, pH 3.8, using a peristaltic pump. The washings were then transferred to a ml volumetric flask and made up to 10 ml with the same buffer and the peptide content was measured by high performance liquid chromatography.

Formulations used in in vivo nasal in vitro assays contain a peptide, inhibitor, 0.1 M phosphate buffer, pH 7.4, purified water, and, if desired, an absorption enhancer. Specific formulations are provided throughout the Examples.

Analytical Methods: For the investigation of leucine-enkephalin and its major metabolites, aliquot samples were obtained.

aliquots are diluted with 200 µL of 0.1 M citric acid, pH 2.3 to stop hydrolysis, and the samples are analyzed (for in vivo in situ tests, washings are injected directly as they are recovered). High performance liquid chromatography

Performed on a 4.6 x 250 mm 0θ column. The mobile phase used was a mixture of 0.1 M NaH ₂ PO ₄ , phosphoric acid and acetonitrile in a ratio of 916: 5: 275 by weight, and the mobile phase flow rate was 1.7 mL / min. UV detection was performed at 210 nm. The retention time of leucine-enkephalin and its major degradation product, Des-Tyr-1eucin-enkephalin, is 7.3 and

4.8 min. The results are expressed as the weight% of leucine enkephalin remaining in the nasal perfusion solution at 100% actual concentration at time 0.

Example 1

For the assay, 10 ml of a 0.1 M phosphate buffer solution, pH 7.4, containing 100 µmol / l of leucine enkephalin as a peptide, was 0.1 µmol / l

3-methyl-1- (4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl) -1-butamine trifluoroacetic acid salt (boron leucine) in the presence or absence of inhibitor. This solution is used for nasal perfusion treatment of the rats described above.

A 10 ml portion of the above peptide solution is placed in a jacketed water-cooled container at 37 ° C.

It is maintained for 11 and is flowed through the nasal cavity of rats using a peristaltic pump. The perfusion solution is led through the nasal cavity, drained through the nostril, transferred to a funnel and returned to the water jacketed container. The perfusion solution was analyzed by the analytical methods described above.

The results of this study are shown in Table I.

• ·

c • f — ι ω Ώ ω r-d

I Ρ Ό JD

C Ρ • rd > χ r-d 1- 1 ω 1 C 9-d Ν • rd ω (D ω Q □ C ω ω ο \ ° (-4 ι

ω Ό Ό Ο

Ν □ _ '{D

C I τΗ

C r-1 • Γ-1 CU Ο 9-4 Ό Ζ3 ω Τ3 ω ω __ι C ί-4 ω ω ο \ ° ι Ε

CZ f-i · Ή

> χ <~ Η ω 1- I ro ^χ ω I C q < "Ο Ν • ι-1 ω ο ω ω jd Ν α ο c Ο. ω (D ^χ ω ο \ ° -1 1

I

ι C C • rd • rd ι-4 ω CU 'CD 9-1 Ο ω ω ro r-d t-L c ro ο \ ° ω ε

Ο ΟΙ μχ χο ΟΙ ΟΙ Γ'Χ UX ο ο ο ο ο ο ο ο +1 +1 +1 +1 +1 +1 +1 +1 ο ΧΟ οχ Γ'Χ <3 · σχ ο Ο ο r-l r Η Γ-1 ΟΙ ΓΟ

ο UX ο Ο- γο UX χο ux ο <d ι ^ χ r-d κχ ΟΙ ΟΙ Γ-Χ +1 +1 +1 +1 -μ +1 +1 +1 ο χο ux <± Γ- 00 CO χο CD χο κχ χο ΟΙ <-4 CO UX ο σχ σχ σχ σχ σχ CO CO

Ο Γ * CD σχ <3 00 σχ rx ο ο ι-1 οι χο UX rx +1 +1 +1 +1 +1 +1 +1 +1 ο Γ- UX ο κχ ο 03 <3 · ο ο- οι rx r-d r-d co r-d οι ΓΟ <± UX ux

CD σχ ux co χο r- r- «-1 • χ • χ ex • χ rx • X • X SHE She t-d Xu She- XO xo She- +1 +1 +1 +1 +1 +1 +1 +1 0 rx ux σχ 0 <3 · 0 ux • X «> ex *> rx * X • x ex 0 <D * 0 σχ X0 rx CD «d 0 σχ σχ r- XO ux ol

SHE UX 0 1-1 0 Ol 0 rx ux 0 XO 0 σχ 1-1 ol ro ux XO r- oo

Without the inhibitor, approximately 58% of the degradation product, Des-Tyr leucine-enkephalin, is formed during the nasal flow of the perfusion solution for 90 minutes. On the other hand, only about 3% of the degradation product is formed under the same conditions in the presence of the inhibitor.

Example 2 ml of 0.1 M pH 7.4 phosphate buffer containing 100 µM leucine enkephalin plus 0.01, 0.03 or 0.1 µM boron leucine inhibitor containing or without inhibitors, as described in Example 1, was used as a nasal perfusion solution in rats. At the end of the assay, the solution is determined by high performance liquid chromatography to determine the degradation products of 1eucine-enkephalin. Our results are shown in Table II. See Table 1.

«· * ·

-H ro

N ^v ro

XD 'ro cd ω' CD c o

CD □ -P rH • h: z)

CD ι — I

C'CD • HC ro

ι- SHE LA She r- THE LA XO LA ι- >> c • χ • χ x * rx <x rx Ε CD t-1 Ή She rh THE r ------ 1 THE 04 04 THE ro Ό CD SHE > E ZD +1 +1 +1 +1 +1 +1 + | +: «Η Ώ CD 'X' E H She XO la <D · r- co CO Xu CD 1 <X <χ ·> ·> • x rx rx «X. + -> _Q rh f-l She Xu THE XO OJ rh CO LA ro 'CD r. Ό She Ox σχ σχ σχ σχ co co · * 0 +> She XD rh ι-I 'CD SHE r-1 C CD CD Ό rh • H CD N Ό 'D r · H q-i E \ CD r- XO 04 <D ' THE <± SHE f-4 • H ι-1 C *> • x ·> ·> rx rx CD CD 'SHE • 1-1 CD i-1 rh 04 rh rh H rh CL ZJ E CD +1 1 ω ZJ D +1 +1 +1 +1 +1 +1 +! -4 ¢ f-1 CD -1 ¢ l rh CD rh xo co THE Γ- LA σχ SHE P FA 1 • χ • χ rx rx ·> • x • X «X Ό SHE -1 ¢ SHE the r-1 CO LA «-1 04 'ZD _D • x Ό She Ox σχ σχ CD CO 00 Γ- E She XD H r ~ H ro CD 4-> H P • H ro rh 4 » 1 r-1 ι-1 e Ό \ SHE LA THE σχ CD She ΧΟ • rH E rh C • x • x  * χ ·> • χ rx rx r-1 ZD Ό • H CD the LA ol CXL OJ rh ro E CD +1 4-H ZJ Z) +1 +1 +1 +1 +1 +; (D rh CD rh CD xo 1 ------ 1 H σχ OJ 04 c CD rh 1 * χ <x • X * χ -x • x CD She ui SHE She σχ σχ σχ xo 1 CD »x Ό CD σχ co Γ- r- XO LA c 'CD She XD r-1 • r-l ω ΓΑ Ώ SHE CD σχ THE oo χο r- θ ' rh CD (-4 • χ ·> • X x * rx <X rx rx j-1 She SHE CD CD rh xo the XD XO θ ' + -> rh +1 P • H : zj +1 +1 +1 +1 +1 +1 +; CD t-1 JD 4-> She • H ι-1 CZD THE LA σχ ο She XO • H »x XZ '(D rx <x • X • x • X • X rx r-1 CD E E CD SHE σχ χο THE She «Η \ 1-1 SHE σχ σχ xo LA <fr 04 rh rh Ό E □ CD CD CD -1 ¢ She LA She She She LA She SHE rh *CD CD rh 04 THE xo σχ XD CL 1-1

The results show that with increasing concentrations of the boron-leucine inhibitor, decomposition of 1eucine-enkephalin is reduced, and higher concentrations of 1eucine-enkephalin are found in the perfusion solution as a function of time.

Example 3

The assay used was 0.1 M phosphate buffer pH 7.4 containing 100 µmol / l leucine enkephalin plus 0.1 µmol / liter boron leucine or 0.1 µmol / liter 4, 4,5,5-Tetramethyl-X- (1-methylethyl) -1,3,2-dioxoborolan-2-methanamine trifluoroacetic acid salt (borovaline) or 0.1 µmol per liter, containing 7,4,4,5,5-pentamethyl-1,3,2-dioxaborolane-2-methanamine trifluoroacetic acid (boron alanine) or without inhibitor.

These solutions were used as a nasal perfusion solution in rats as described in Example 1. The degradation products of leucine-enkephalin were assayed by HPLC as described in Example 1. Our results are shown in Annex III. See Table 1.

100 µmol / liter 1eucine-enkephalin in situ nasal degradation 10 ml nasal perfusion

She She rh CXJ <-1 <- r · • X «X rx rx ·> <X • χ CXJ She rx (XJ CXJ rx <± XO (d +1 +1 +1 +1 +1 +1 +1 +1 She σχ l / X r-l UX σχ • X rx ·> • X rx • X *> rx She r-l co <fr r * σχ Xu She σχ σχ 00 oo r- χο ux

She σχ ux XO ux ux -X She ι-1 <-1 ι-1 ι-1 CXJ CXJ +1 +1 +1 +1 +1 +1 +1 +1 She rx Γ- co XO rh CXJ ΓΧ She XO UX rh σχ xo oo She σχ σχ σχ σχ 00 CD r-

She ux She r * rx ux χο ux • X * X «X r> r <x • X She rh rx rh rx CXJ CXJ rx +1 +1 +1 +1 +1 +1 +1 +1 She Xu UX r- 00 co XO rs <x rx • X • X • X rx r. She XO rx XO CXL «-Η 00 UX She σχ σχ σχ σχ σχ oo CD

She σχ ux 00 XO r- r- <-1 She She rh XO r- xo XO Γ ' +1 +1 +1 4-1 +1 +1 4-1 4-1 She rx UX σχ She <fr SHE XO She <± She σχ χο rx She ι-1 She σχ σχ r- XO ux CXJ

She ux She She She ux She She rh CXJ rx XO σχ

The table compares the inhibitory effect of equimolar concentrations of three different ¢ 4-aminoboronic acid derivatives on the degradation of leucine-enkephalin. Boron leucine and boral valine inhibitors are more effective than boron alanine.

Example 4

0.1 M phosphate buffer solution, pH 7.4, containing 100 µM leucine-enkephalin, in addition to known inhibitors such as 10 µM bestatin or 100 µM puromycin, and 0 as the tested inhibitor. Boron leucine (1 µmol / L) was used as a nasal perfusion solution in rats as described in Example 1 and the degradation products were also assayed by high performance liquid chromatography as described in Example 1.

The results of the study are presented in Annex IV. Table 1.

c ω _D -Η ro TD r ~ Η

Ο

C • Γ — I ω ε ο

ZJ Q.

ι-1 ο σχ ο Γ- Γ * Χ σχ χο Lix C ·> ·> • χ • X ex • X <χ γ-I • ι-1 ο Γ · Η γα Γ-1 ΓΛ OJ οι γλ Ό ω ε ZJ +1 +1 +1 +1 +1 +1 +1 +1 Σ3 ω . γ-Η Ο ΧΟ UX <± Γ- CO 00 Γ- 1 ο ex ex • χ <χ • χ • χ • χ ι-1 Ν ο ΧΟ Γ ^ Χ ΧΟ CXJ ι-I 00 σχ • χ Ό ο σχ σχ σχ σχ σχ οο οο ο η Γ-1 γ-1 ο <± «~ Ι οι OJ χο Γ- ex ** ex ex <χ • X • χ • χ r-1 C Ο OJ Γ ^ Χ <fr rx OJ σχ χο Ό • rH Ε Ο +1 +1 +1 +1 +1 +1 +1 +1 Ζ3 >> Ε ο Γ- ' Γ'Χ <Γ σχ χο σχ σχ Ο e \ ex ♦ X • X ex «χ <χ ex Ο C ι ο ΧΟ γ- | OJ 00 ΓΧ Γ'Χ 00 Ο ο σχ σχ 00 * γ - · Γ * χο <± <-1 ο_ Γ-I ο ΧΟ Γ- rx CD Γ ^ Χ Γ- οο <-1 <Χ ** ex • χ ex <χ ex • χ ο r-H r | Γ'Χ rx σχ χο 00 r-1 C Ό • (Η +1 +1 +1 +1 +1 +1 +1 +1 Ε Ή CU ο Γ-Χ r- ο Xu ο r * x rx \ + -> tn ο r * ο χο σχ ΟΙ r- ο CD ο σχ σχ σχ co r- Γ- σχ t-ι .Q t-1 ο σχ UX CO STONE ο- ο »χ • X • X • X • X * χ ex ο Ο <-ι ΧΟ Γ- χο χο Γ- ο -Η Η-ι +1 +1 +1 +1 +1 +1 +1 +1 • γΗ : zj .D ο OJ σχ σχ ο ο UX ·· -1 ι-1 ex • X ο • X x * • X • χ * χ JZ 'CD ο ο σχ χο Γ ^ Χ Ο γ- ( C C ο σχ σχ Γ- χο σχ 04 1-1 ι- ^- 1 ω t-L ο υχ ο ο ο σχ ο Ο ω γ- CXI Γ'Χ χο σχ

Ο Q.

• · • · " · ···· ·· ·the

The table above compares the in situ degradation of the leucine-enkephalin peptide without the use of an inhibitor and in the presence of three different inhibitors administered at different concentrations. The order of inhibition of degradation for the various inhibitors is as follows: boron leucine) bestatin / puromycin. The inhibitory effect of boron-leucine is better than that of bestatin, although its concentration is only one-hundredth of the latter. This clearly demonstrates the significant potency of boron-leucine over known inhibitors.

Example 5

Phosphate buffer containing 0.1 mol / l, pH 7.4, 35 mol / l

LH-RH (Fragments 3-10) / is used in the absence of an inhibitor or in the presence of 0.46 µmol / L of a boron leucine inhibitor to treat rats as described in Example 1. The perfusion solution was subjected to high performance liquid chromatography as described in Example 1, except that the mobile phase was a mixture of 0.1 mol / L Ν3Η2ΡO:: H POO ^: CH ^ CN (916: 5: 240). ), the mobile phase flow rate was 1.7 ml / min and the analysis was performed at 210 nm under UV. Our results are shown in Table V.

• ·

- 20 V. Table Yumol / L LH-RH (Fragments 3-10) disappearance in nasal perfusion solution (10 mL) in the presence or absence of 0.46 µmol / L boron leucine

Time Boron-leucine 0.46 / µmol / l (minute) without boron-leucine 0 100.0 100.0 5 96.0 96.7 10 93.8 97.5 20 91.6 95.5 30 85.3 96.5 45 70.4 95.8 60 61.3 91.9 90 45.0 92.9 120 24.4 86.4

The disappearance of LH-RH is significantly faster in the absence of the inhibitor than in the presence of the inhibitor. The presence of boron-leucine at very low concentrations results in a% retention of the compound while only 24% remains without the inhibitor.

Example 6

100 µM Tyr-Phe-Met-Arg-Phe-NH ₂ containing 0.1 M phosphate buffer pH 7.4 was administered nasally to rats as described in Example 1.

·· ♦ · · · · · · ··· · · · · · · · · · ·

The peptide is used in the absence of inhibitor or in the presence of 2 µM boron leucine inhibitor. The perfusion solution was subjected to high performance liquid chromatography as described in Example 1, except that the mobile phase was a solution of 0.1M NaH 2 PO 4: H 2 PO 4: CH 2 CN (200: 1). 60) and that the mobile phase is used at a flow rate of 1.4 ml / min. Detection was performed at 210 nm under UV.

Results are reported in Annex VI. See Table 1.

VI. Spreadsheet

Nasal disappearance of 100 µmol / L Tyr-Phe-Arg-Phe-NP NP in situ in a nasal perfusion solution (10 mL) without inhibitor and in the presence of a Yumol / L boron leucine inhibitor

Time (minute) Boron-leucine without 2 / µmol / liter boron leucine 0 100.0 100.0 5 85.4 93.8 10 80.7 20 71.1 84.3 30 59.5 78.8 45 44.4 70.5 60 30.4 63.3 90 10.6 49.5 120 2.8 32.9

• ·

The results show that 33% of the peptide remains intact while using 2 µmol / L of boron leucine inhibitor and only 3% without inhibitor.

Example 7

per rat

100 µg

0.1 mol / L phosphate buffer, pH 7.4, containing 1eucine enkephalin, was administered to rats using 0.05% sodium cholate penetration enhancer. Peptide and penetration and enhancer without inhibitor / boron leucine inhibitor

Administered at a dose of 0.054 µg / rat. The test model is the same as in Example 1, except for the solution to be tested and the sampling procedure. The administration is carried out by adding 50 ul of a peptide solution containing or without an inhibitor to one of the rat nostrils. The peptide solution was added to a 50 µL Hamilton syringe with a 1/2 cm PE-50 tube attached to its end. At intervals (at one time in a rat), the drug was flushed through the other nostril using 10 mL of 0.1 M pH 3.8 buffer using a peristaltic pump. The nasal wash solution is placed in a 10 ml volumetric flask, made up to 10 ml with the same buffer and the peptide is added to the

Analyze as described in Example 1.

The results obtained are shown in Table VII. Table 4 below.

• ···

f-l t-L ο >> -Ρ ι-1 Κ- • ι-1 :She ι _Q N • ι-1 ι ^- 1 ω Σ £ _ CD α c C

SHE c • H r-1 Chromatography of the residue ·· -1 : = J JD J = • 1-1 r-l CD _c 'CD c C

r * r-l She She <-1 She +1 +1 +1 +1 xo XO OX CXL She r-l Γ-I CM

CM CO r * · <—ι

LA

+1 +1 +1 IA r-l <± CTX r * Xu

XO

oxy 03 * χ She LA SHE r-H «-1

+1 +1 +1 «-1 co rx • X «χ 00 co 0x1 ΓΑ CM r ~ d

r- CXL «-1 CD CXL rx rx • X • X rx CM r-l γα CM +1 +1 +1 +1 +1 She LA She LA 0X rx rx rx rx rx Γ- ! ------ 1 r-l Xu Γ- r-l 0x1 0x1 «-1

Ox CXJ CM CM CM • X rx rx rx rx rx XO LA σχ σχ XO t-1 +1 +1 +1 +1 +1 +1 r- She THE- co XO THE rx <x rx »x • x rx CXJ • cf σχ r- CM σχ LA THE r-l

SHE LA SHE She SHE SHE r-l CM THE LA

· · · · · · · · · · · · · · · · ·

- We monitor the disappearance of 24 Leucine-enkephalin in vivo, in situ, and the appearance of its largest metatarsus, Dez-Tyr-1eucin enkephalin. Without an inhibitor, the pathways for the disappearance of leucine-enkephalin are the absorption and degradation to des-Tyr-leucine-enkephalin, as well as the absorption to the latter. While the disappearance of 1eucine-enkephalin in the presence of an inhibitor is primarily attributable to absorption, the observed degradation is negligible.

Example 8

The procedure described in Example 7 was followed, but the Tyr-Phe-Met-Arg-Phe-N1 · 2 peptide was tested. The peptide is administered at a concentration of 50 µg / ml, 50 µl titer / rat, with or without the addition of a boron leucine inhibitor. The inhibitor was used at a concentration of 0.3 µg / rat. No penetration enhancer is used in this assay.

Our results are shown in Table VIII. See Table 1.

VIII. Spreadsheet

Tyr-Phe-Met-Arg-Phe-NHg (50 in vivo nasal disappearance in the presence of 0.3 or / ug / rat) in situ, / ug / rat boron leucine distant food

Time (minutes)

Boron-leucine

0.3 µg / rat without boron leucine

0.0 100.0 + 0.0 100.0 + 0.0 2.5 60.4 + 8.5 84.2 + 3.8 5.0 39.9 + 5.2 67.3 +. i> ⁷ 10.0 21.5+ 3.9 42.1 + 6.7 The results it show that Tyr-Phe-I -Arg-Phe-NHG disappearance faster in the absence of inhibitor

Met

Claims (9)

Claims CLAIMS 1. A process for the preparation of protein or peptide containing stable pharmaceutical compositions using pharmaceutically acceptable carriers and a stabilizing system, comprising the steps of: a stabilizing system comprising an ού-aminoboronic acid derivative of the formula - HW stands for mineral acid. sulfonic acid, alkanoic acid or perfluoroalkanoic acid; - Y is a group of a dihydroxy compound having at least two hydroxy groups separated by at least two linked chain or ring atoms from the carbon atoms of said chain or ring and optionally from a nitrogen, sulfur or oxygen heteroatom They are, - R is alkyl or -Ch ^ R ¹ ; - R is -XR, aryl or aryl substituted with one or more alkyl groups; - X is oxygen or sulfur; R is hydrogen, alkyl or -SiR ³ R ⁴ R ⁵ ; - R ^3, R ⁴ and R ³ is independently alkyl, aryl or one or more alkoxy-substituted aryl. The process according to claim 1, characterized in that for hydrolysis by proteases. • · * «** 27 sensitive proteins or peptides are used. 3. The method of claim 2, wherein the protein or peptide is selected from the group consisting of leucine-enkephalin, LH-RH, Tyr-Phe-Met-Arg-Phe-N1 ^, Tyr-Bradykinin, and Thymopoietin? t. A process according to claim 1, wherein Y is a substituent (CH-) _? CC (CH ₃ ) _? . I I -CH2CH2-, (CF j) ₂ CC (CF j) 2, or - (C ₂ Η) Ν H (CH ₂₎ a compound of formula (I) wherein ~ group is used. 5. A process according to claim 1 wherein R is -CH 1, -CH 1 -CH 2 -, -Ch 2 Cl 2 - (CH 1) 2, or a benzyl group. 6. A process according to claim 1 wherein HW is a compound of formula (I) containing CF 2 CO 2 H, CH 2 Cl 2 H, HCl or HBr. The process according to claim 1, wherein the N-amino-boronic acid derivative is 3-methyl-1- (4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl). ) -1-butamine trifluoroacetic acid salt. 8. The method of claim 1, wherein the oral dosage form is formulated for oral administration. • · · · • · «· · • 4 *« «· * ♦ ·» ··· 9. The method of claim 1, wherein the dosage form is for nasal administration.